Antigenicity of human melanoma cells transfected to express the B7-1 co-stimulatory molecule (CD80) varies with the level of B7-1 expression

 The aim of this study was to compare the antigenicity of human melanoma cells molecularly modified by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodified melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%–28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%–90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21-B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony-stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21-B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-mediated cytotoxicity against unmodified melanoma cells by PBMC co-cultured for 5 days with the modified or unmodified melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modified cells in proportion to their level of B7-1 expression. Received: 14 October 1999 / Accepted: 3 December 1999     

Buried charged surface in proteins

BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Characterization of Genes Involved in Biosynthesis of a Novel Antibiotic from Burkholderia cepacia BC11 and Their Role in Biological Control of Rhizoctonia solani

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.     

Characterizing a hybrid zone between a cryptic species pair of freshwater snails

Characterizing hybrid zones and their dynamics is a central goal in evolutionary biology, but this is particularly challenging for morphologically cryptic species. The lack of conspicuous divergence between parental types means intermediate hybrid forms often go undetected. We aimed to detect and characterize a suspected hybrid zone between a pair of morphologically cryptic lineages of the freshwater snail, Radix. We sampled Radix from across a contact zone between two mitochondrial lineages (Radix balthica and an undescribed lineage termed ‘MOTU3’) and detected admixture between two nuclear genotype clusters, which were significantly but not categorically associated with the mitochondrial lineages. Using a model selection approach, we show that the admixture cline is best explained by an interaction between precipitation and temperature gradients over the area, rather than geographic distance. We thus hypothesize that the correlation with climatic gradients suggests environmental selection has played a role in maintaining the hybrid zone. In a 2050 climate change scenario, we furthermore predict an expansion of one of the nuclear clusters and a widening of the hybrid zone as the climate warms and dries.     

The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.     

The multifaceted role of oestrogen in enhancing Chlamydia trachomatis infection in polarized human endometrial epithelial cells

The oestrogen receptor (ER) α-β+ HEC-1B and the ERα+β+ Ishikawa (IK) cell lines were investigated to dissect the effects of oestrogen exposure on several parameters of Chlamydia trachomatis infection. Antibody blockage of ERα or ERβ alone or simultaneously significantly decreased C. trachomatis infectivity (45-68%). Addition of the ERβ antagonist, tamoxifen, to IK or HEC-1B prior to or after chlamydial infection caused a 30-90% decrease in infectivity, the latter due to disrupted eukaryotic organelles. In vivo, endometrial glandular epithelial cells are stimulated by hormonally influenced stromal signals. Accordingly, chlamydial infectivity was significantly increased by 27% and 21% in IK and HEC-1B cells co-cultured with SHT-290 stromal cells exposed to oestrogen. Endometrial stromal cell/epithelial cell co-culture revealed indirect effects of oestrogen on phosphorylation of extracellular signal-regulated kinase and calcium-dependant phospholipase A2 and significantly increased production of interleukin (IL)-8 and IL-6 in both uninfected and chlamydiae-infected epithelial cells. These results indicate that oestrogen and its receptors play multiple roles in chlamydial infection: (i) membrane oestrogen receptors (mERs) aid in chlamydial entry into host cells, and (ii) mER signalling may contribute to inclusion development during infection. Additionally, enhancement of chlamydial infection is affected by hormonally influenced stromal signals in conjunction with direct oestrogen stimulation of the human epithelia.